Presentation Details
| Unraveling the Anticoagulant Functions of FV Using Monoclonal Antibodies Sean Quinn1, Francis Ayombil1, Matthew Bunce1, Rodney M.Camire1, 2. 1Department of Pediatrics, The Raymond G.Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.2The University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA |
Abstract
BACKGROUND: Coagulation factor V (FV) has both pro- and anti-coagulant functions. As a key component of prothrombinase, activated FV (FVa) enhances the relative rate of thrombin generation by 300,000-fold compared to FXa alone. While the procoagulant functions of FV have been extensively studied, its anticoagulant functions are less understood. Together with protein S (PS), FV is thought to function as a cofactor for activated protein C (APC) and TFPIα in the inactivation of FVIIIa and FXa, respectively. Interestingly, FVa does not show these anticoagulant properties. OBJECTIVE: Here, we generated FV-specific monoclonal antibodies (mAbs) as tools to better understand FV function in these anticoagulant pathways and their procoagulant response. METHODS: Procoagulant FV-specific mAbs (GB5, IC1, 3H8, and 6C2) were identified from mice immunized with human FV. Kinetic binding studies using biolayer interferometry (BLI) were used to determine the affinities for antibody binding to physiologic forms of FV including FV, FV-short, and FVa. Immunoblotting, BLI binning, and peptide arrays were used to define the binding epitopes for our FV-specific mAbs. The hemostatic function of each mAb was assessed in tissue factor-initiated thrombin generation assays (TGAs) using pooled normal, or hemophilia A plasmas. The mechanism and effect of mAbs on APC and TFPI anticoagulant functions were evaluated via both plasma-based TGAs using PS-depleted plasma (PS-DP) and TFPI-depleted plasma (TFPI-DP) and in purified component assays (i.e. prothrombinase). RESULTS: All four mAbs bound physiologic forms of FV with sub-nanomolar affinities. Immunoblotting data confirmed that GB5 binds the light chain of FV. Further, GB5 blocked plasmin-mediated cleavage of FV light chain, suggesting its epitope is proximal to the known plasmin cleavage site at R1765. Additional experiments revealed that while IC1, 3H8, and 6C2 have distinct peptide sequences, they compete for binding to FVa which indicates that they have overlapping binding epitopes. In TGAs using pooled normal or hemophilia A plasmas, each mAb modestly enhanced thrombin generation. In the presence of added APC, 6C2 and GB5 completely restored thrombin generation, whereas IC1 and 3H8 partially restored thrombin generation. Purified component assays showed that the mAbs did not enhance thrombin activation of FV or FVa cofactor activity in prothrombinase. These results demonstrated that all four mAbs may alter APC anticoagulant functions in plasma with a procoagulant outcome. In TGAs using TFPI-DP, none of the mAbs altered thrombin generation suggesting that they may impact the TFPIa pathway. In addition, the effect of GB5 on thrombin generation was abolished in PS-DP. Together, these results show that the mAbs disrupt the APC and TFPIα anticoagulant functions of FV. In addition, GB5 disrupts FV anticoagulant function with PS. CONCLUSION: In summary, we have identified four distinct FV-specific mAbs which alter the APC and TFPIα pathways and are useful tools to better investigate the anticoagulant properties of FV. These antibodies may be useful for rebalancing coagulation via FV’s anticoagulant functions.
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No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.